Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.

Abstract

Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(a-[3S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.Both major rapid DNA sequence determination methods (1, 2) rely on thin denaturing polyacrylamide gels (3) to fractionate single-stranded DNA reaction products. To determine a sufficient length of sequence data from an experiment, it is usual to load aliquots from a reaction onto two or more gels run for various times. It would be advantageous to be able to obtain more sequence from each gel because this would allow either more total sequence to be obtained or fewer gels to be run per reaction. We describe two methods for increasing the amount of readable sequence per gel and demonstrate their effectiveness with the dideoxy sequence analysis technique.One of the main limits on the length of sequence obtainable from an autoradiograph is the progressively poor separation of the bands corresponding to longer DNA molecules. Yet the spacing between the shorter DNA molecules is wider than is required for correct interpretation of the sequence pattern. It is possible, by use of a suitable gradient positioned only in the lower (anode) end of a gel, to selectively reduce the spacing between these shorter DNA molecules. By virtue of having traveled through a greater length of polyacrylamide gel, the higher molecular weight DNA molecules, lying above the gradient, will have an increased separation. We have found that an effective gradient consists of an increase in Tris/borate/EDTA (TBE) buffer concentration towards the bottom of the gel. A limit on the use of gradient gels has been the inconvenience and length of time needed for pouring such gels. We present a rapid technique, using simple apparatus, for pouring gradient gels, making their routine use for DNA sequence analysis realistic.The principle by which a buffer gradient can be used to reduce the vertical band spacing on a polyacrylamide sequencing gel is that, as the buffer concentration increases progressively in the lower 10-15 cm of the gel, electrical resistance per cm down the gel decreases. This is because the buffer is the major charge carrier in the gel. Because the current is constant throughout the length of the gel, by Ohm's law the voltage drop per unit length will also decrease towards the anode. It is the potential difference across the gel that drives the polynucleotide migration, and so a fall in voltage drop per cm will cause a reduction in band migration rate. Thus the spacing between DNA molecules of n and n + 1 nucleotide residues can be reduced as they enter a gradient of increasing buffer concentratio...

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

“…This vector is identical to pSK(-) except that it contains a HincII-EcoRV deletion in the polylinker, making it useful for the gene disruptions described below. Single-stranded DNA templates were prepared from the resulting clones and used for DNA sequence analysis as described previously (Sanger et al, 1977;Biggin et al, 1983). The 5'-terminal non-coding sequences of CKI2 were determined from the 2.5-kb genomic clone by the dideoxy chain termination method applied to denatured doubleMolecular Biology of the Cellstranded DNA (Sambrook et al, 1990).…”

Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

“…This and similar analysis of subcloned fragments revealed that most of the insert in CP1 was required for complementation. The entire -7.0-kb insert in CP1 was sequenced by using the dideoxynucleotide chain termination method (Sanger et al, 1977) modified for the use of 35S-dATP (Biggin et al, 1983) and T7 polymerase (Sequenase; USB, Cleveland, OH). A series of unidirectionally deleted templates for sequencing was generated by transferring restriction fragments isolated from the insert in CP1 into the vector pVZ1 and employing the exonuclease III method (Henikoff, 1987).…”

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.

“…We determined the nucleotide sequences of the cDNAs encoding Xenopus LDH-A, LDH-B, and LDH-C, pig LDH-A and LDH-B, and rat LDH-B and LDH-C. 11 We have used these seven LDH sequences deduced from their cDNA sequences and 22 other published LDH sequences, plus three unpublished fish LDH-A sequences, to analyze the evolutionary relationships ofLDH isozymes from mammals, birds, an amphibian, fish, barley, and bacteria.…”